ABSTRACT Sickle cell anemia is one of mankind's most common hereditary monogenic diseases and a global health burden. A therapeutic goal is to increase fetal hemoglobin concentration to protect erythrocytes from sickle polymer-induced injury. Fetal hemoglobin gene expression is regulated by cis-and trans-acting loci. Although the trans-acting loci have been intensively studied and BCL11A is being targeted genetically, less attention has been paid to cis-acting regulators, which in some instances might be of paramount importance. Herein we focus on novel cis-acting loci that we found to be associated with elevated fetal hemoglobin in the Arab-Indian haplotype of sickle cell anemia. Our central hypothesis is: Variants in the ?-globin gene cluster locus control region in sickle cell anemia with the Arab-Indian haplotype modulate fetal hemoglobin gene expression by differential looping of the locus control region to globin gene promoters and account, in part, for the variance in fetal hemoglobin levels associated with different HBB haplotypes. We will use an induced pluripotent stem cell (iPSC)-based system capable of recapitulating both fetal and adult-type blood cell production that provides a flexible model for mechanistic studies to validate this hypothesis. We propose three specific aims: Aim 1: Genetically modify sickle cell disease haplotype-specific iPSCs to assess the effects of putative cis-acting regulators of fetal hemoglobin expression. We will use gene editing to target selected cis-acting elements to modulate fetal hemoglobin gene expression in adult-type erythroblasts derived from sickle cell anemia patients with the high Arab-Indian and low Benin fetal hemoglobin HBB gene cluster haplotypes. Aim 2: Functionally validate novel cis-acting fetal hemoglobin modulators using the directed differentiation of sickle cell disease-specific iPSCs into fetal and adult-type blood cells. Directed differentiation of sickle iPSCs into fetal and adult hemoglobin-expressing erythroblasts will allow us to explore the cell intrinsic effects of modified regulatory regions. Aim 3: Dissect the effects of cis-activating fetal hemoglobin modulators in iPSC-derived erythroid cells using chromosome conformation capture, chromatin immunoprecipitation, and global transcriptional analyses. We will define the mechanism of cis-acting regulation through assays examining the effects of engineered changes on the expression of globin genes, fetal hemoglobin synthesis and chromosomal looping. The proposed studies use both established and novel systems and strategies to: 1) evaluate the role of novel, cis-acting fetal hemoglobin modulators in iPSC-derived erythroid cells, 2) reveal the basic biology behind globin switching during blood cell development, and 3) lay the groundwork for the development and testing of novel, patient-specific therapeutics. These transdisciplinary studies will be carried out in the complementing laboratories of three investigators with expertise in stem cell biology (Murphy), sickle cell disease and globin gene regulation (Steinberg), and chromatin dynamics (Dai).